The contractile mechanism of vertebrate striated muscle is activated by the binding of calcium to troponin. Recent studies have indicated that there are two classes of calcium-binding sites and that binding to both of them is necessary for contraction to occur. In addition it has been shown that the affinity of calcium for troponin is increased when myosin is linked to actin as in rigor. Certain dynamic properties of living muscle, such as the effect of tension and length on activation, could be accounted for if it can be shown that the affinity of calcium for troponin is influenced by variables such as cross-bridge attachment, sarcomere length, and shortening. The aim of this investigation will be to determine how the binding of calcium to isolated myofibrils and glycerol-extracted muscle fibers is influenced by perturbations in the above parameters. There are now available a number of ATP analogs which can "freeze" the contractile system into various modes of cross-bridge orientation and attachment that are believed to represent stages of the normal cross-bridge cycle. These modes have been reasonably well defined biochemically and structurally. By studying how calcium-troponin affinity is affected by such analogs, as well as by changes in tension, sarcomere length and shortening velocity, it should be possible to draw some conclusions about how calcium mobilization and binding are controlled by mechanical conditions during the contraction-relaxation cycle of living muscle.